It has been proposed that rare, treatment-resistant neoplastic stem cells are critical for long-term tumor growth. Normal stem cells require signals from a stromal niche to self-renew. However, the role of such niche-signals in regulating cancer stem cells (CSCs) is largely unknown. The Notch signaling pathway regulates neural stem cell self-renewal, and recent studies found that CSCs in glioblastoma (GBM) have elevated Notch activity. Our recent data shows that Notch pathway inhibition by gamma-secretase inhibitors reduced CD133 expression and reduces glioblastoma neurosphere propagation both in vitro and in vivo. In the current application, we propose using GBM neurosphere cultures to determine the mechanism and niche-dependence of Notch activation in CSCs. Specifically, we will seek to determine if Notch activation is driven by juxtacrine signaling between tumor cells replicating the classical process of lateral inhibition, by stromal niche signals, or both. Aim1 - Determine if activation of Notch in CSCs is mediated by juxtacrine signaling via Notch ligands expressed in better-differentiated tumor cells. Aim2 - Define if the endothelial cells function as CSC niche by providing Notch ligands to CSCs in glioblastoma. Aim3 - Determine if regulation of Notch activity in endothelial cells contributes to the non-functional microvascular proliferation in GBM. Answering these questions will facilitate the development of novel therapies and enhance our understanding of cancer stem cell biology.